The invention relates to oligonucleotides for the detection and visualization of bacteria belonging to the genomic species Escherichia coli in a sample. More particularly, it relates to an oligonucleotide capable of hybridizing specifically with the ribosomal RNA (rRNA) or to the corresponding gene (rDNA) of the genomic species Escherichia coli (including Shigellae with the exception of S. boydii serotype 13)/Escherichia fergusonii. 
It likewise relates to a procedure for detection of the genomic species in question employing this oligonucleotide as well as the use of said oligonucleotide in a gene amplification procedure.
In this document, the term xe2x80x9cEscherichia colixe2x80x9d (E. coli) denotes the genomic species (genomospecies) containing the strain-type Escherichia coli ATCC 11775 (=CIP 58-8). A genomic species is a collection of strains whose deoxyribonucleic acid (DNA) has a homology of more than 70% with the DNA of the strain-type of the species considered with a thermal instability of the hybridized DNA of lower than 5xc2x00 C. (Grimont, 1988; Wayne et al., 1987). Following these criteria, the genomic species E. coli includes, apart from the strains usually identified as E. coli, the strains traditionally classed as Shigella (S. dysenteriae, S. flexneri, S. boydii, S. sonnei) with the exception of the serotype 13 of S. boydii (Brenner et al., 1973). In strictly applying these criteria, it is possible to argue that Escherichia fergusonii belongs to the genomic species E. coli (Farmer et al., 1985).
E. coli is usually a commensal bacterium of the colon of man and of warm-blooded animals. For this reason, its presence in a sample of water, of food, or from the environment, is interpreted as an indication of fecal contamination (indicative bacterium). Thus, an alimentary product must not contain more than a certain number of living cells of E. coli (being able to form a colony on a solid culture medium) in a defined mass of product (these numbers vary according to the product). For example, drinking water must not contain any living cell of E. coli in 100 ml (De Zuane, 1997). The counting of the E. coli is essential in order to estimate the hygienic quality of a food.
Strains of the genomic species E. coli can be pathogenic. Among these strains is found any which is commonly called Shigella, the agent of bacillary human dysentery. The strains commonly called E. coli can cause different infections in man or in animals according to the provision with pathogenic genes (urinary infections, choleriform or hemorrhagic diarrhea, dysentery syndrome, hemolytic and uremic syndrome, septicemia, neonatal meningitis, various purulent infections).
The identification of a strain of the genomic species E. coli (taxonomic identification) is important in order to question or demonstrate the fecal contamination of water or food. It is likewise important in the case where the bacterium is isolated in a normally sterile or almost sterile biological medium (urine, blood, cerebrospinal fluid, collection of fluid in a tissue or in a closed space of the body). In the open spaces of the body (digestive tract) or the feces, the presence of E. coli is commonplace and the identification of pathogenic factors of E. coli is of paramount importance in the taxonomic identification.
The taxonomic identification of E. coli is conventionally based on the isolation and the culture of the bacterium on a solid gelatinous medium and the application of some biochemical tests. The appearance of colonies on a gelatinous medium requires at least 18 hours. In the case of samples from the environment, culture for some days is often necessary in order that all the colonies which ought to develop appear. The application of biochemical tests starting from an isolated colony again requires 18 to 48 hours. By way of example, the counting of E. coli in water necessitates the filtration of a volume of water through a sterile membrane, the placing of the membrane on a semi-selective and/or indicator medium, incubation (48 hours) allowing colonies of a characteristic (but not absolutely specific) color to develop, which are then counted. As each isolated colony is supposed to be derived from a bacterial cell, the counting of the E. coli by volume units can be carried out. It is wise to check that the isolated colonies indeed correspond to the species E. coli and this requires at least 18 hours more.
Recently, techniques based on the detection of specific nucleotide sequences of the genomic species E. coli have been described. Thus, the detection by gene amplification (PCR type) of the gene encoding beta-glucuronidase allows the presence of E. coli in a sample to be identified. This method is especially used qualitatively and the interpretation of the gene amplification is frequently hampered by the possibility of contamination due to the dispersion on the apparatus and experimental tools of some nucleic acid fragments.
In situ hybridization is an interesting alternative in gene amplification. An oligonucleotide probe which is labeled (generally by a fluorescent substance) penetrates into the previously treated bacterial cells in order to facilitate this step. According to whether the ribosomal nucleic acids have or do not have a complementary (target) sequence to the probe, the probe will fix to its target and will not be removed by washing. The bacteria having retained the probe in this way become labeled (for example fluorescent) and visible by microscopic examination.
The ribosomal ribonucleic acids (rRNA) form the preferred target in hybridization in situ because of the number of copies per cell (10,000 to 30,000), which is higher than the number of copies of messenger RNA after induction (100 to 200) or of a given gene (one to several). These ribosomal RNAs (rRNAs) are identified according to their sedimentation constant (for the bacteria: 5S, 16S and 23S), present in the small subunit (16S rRNA) or the large subunit (23S and 5S RNA) of the ribosome.
The largest rRNAs are the 16S (approximately 1500 nucleotides) and the 23S (approximately 3000 nucleotides). A complementary nucleic probe of a part of an rRNA would be able to hybridize with this rRNA but also with the complementary strand of the gene (rDNA) which has encoded this rRNA. Various applications of this methodology have been published (Amann et al., 1990; DeLong et al., 1989; Giovannoni et al., 1988; Trebesius et al., 1994).
These rRNAs in fact appeared as the most appropriate molecules to serve as a molecular chronometer in the evolution of bacteria (Brenner et al., 1969; Doi and Iragashi, 1965; Moore and McCarthy, 1967; Pace and Campbell, 1971; Takahashi et al., 1967). The primary structure (sequence) of the rRNAs contains highly conserved regions and others which are hypervariable (Sogin et al., 1972; Woese et al., 1975). The perfection of a DNA-rRNA hybridization method (Gillespie and Spiegelman, 1965) has been followed by a very large number of publications applying this approach to the taxonomy and the phylogeny of bacteria and to the identification of badly classified bacteria (Johnson et al., 1970; Palleroni et al., 1973; De Smedt and De Ley, 1977).
Generally speaking, in a hybridization experiment bringing into play given sequences, the result depends greatly on the temperature and on the molarity of sodium ions of the reaction medium. For a reaction medium of given composition, an optimal hybridization temperature is defined. If the temperature is increased, the reassociated strands will finish by separating. The temperature necessary for this separation depends on the length of the perfectly hybridized (apparently perfect) part of the sequence and on its nucleotide composition. A temperature only allowing hybridization of the longest sequences is called restrictive (in opposition to optimal). Mispairings during hybridization make the thermal stability of the hybridized molecules fall.
The specificity of the hybridization in situ will therefore depend on the quality of the probe capable of recognizing and of hybridizing with a complementary sequence present in an rRNA.
Kohne et al. (1968) described a method for preparing probes reacting with the rRNA without, however, indicating how to detect E. coli specifically.
Gxc3x6bel and Stanbridge (1984) use a cloned rDNA gene for detecting mycoplasmas contaminating tissue cultures.
Galpin et al. (1981) used the hybridization of genes encoding rRNA to detect infections with Mycoplasma pulmonis in mice.
U.S. Pat. No. 4,851,330 describes a strategy for obtaining nucleic acid fragments which can be used as a probe reacting with the rRNAs.
WO-A-84/02721 describes methods for detecting the microorganisms infecting a human or animal body, by using probes which hybridize with the rRNA. It is not indicated how to detect or identify E. coli. 
Berent et al. (1985) show the interest in oligonucleotide probes in relation to cloned probes.
French Patent 2 596 774 proposes the use of a complementary oligonucleotide of the bacterial rRNA as a probe and describes two universal oligonucleotide probes.
Gxc3x6bel et al. (1987) use a synthetic oligonucleotide reacting with the rRNA or its gene with the aim of identifying mycoplasmas.
U.S. Pat. No. 5,084,565 describes a so-called specific oligonucleotide probe of E. coli. For a target, this probe has the nucleotide zone 465 to 477 (numbering of the nucleotides according to Brosius et al. [1978]).
The probe would react with Escherichia fergusonii and Shigella boydii serotype 13 (in addition to E. coli and Shigella) and would not react with Citrobacter koseri. Nothing is said as to the reaction of this probe with the species of the genus Cedecea which are phylogenetically close to E. coli. 
U.S. Pat. No. 5,593,841 mentions a probe reacting with the 995-1030 region of the 16S rRNA of E. coli. This probe reacts with E. fergusonii but does not react with any of the strains of E. coli tested and does not react with Shigella dysenteriae. Nothing is said as to the reaction of this probe with Citrobacter koseri (=C. diversus) and the species of the genus Cedecea which are phylogenetically close to E. coli. 
Kwok et al. (1990) showed that a mispairing at the level of the 3xe2x80x2 end of a primer used in gene amplification (PCR) affects the efficacy of the amplification.
Cha et al. (1992) have described a test called xe2x80x9cMismatch Amplification Mutation Assayxe2x80x9d in which a primer shows a mispairing with the target sequence of a mutation to be detected, and two mispairings with the corresponding sequence of the wild allele. These mispairings concern the 3xe2x80x2 end part of the primer. Under these conditions, their PCR system only detects the mutant allele. This method has been applied to the specific detection of Salmonella enterica serotype Enteritidis (Lampel et al., 1996) by creating a mispairing at the penultimate position of the 3xe2x80x2 end of a primer.
The invention proposes an oligonucleotide for the detection and the specific and rapid visualization of bacteria belonging to the genomic species Escherichia coli in a sample. It therefore relates to an oligonucleotide capable of carrying out a specific hybridization with the genomic species of Escherichia coli (that is to say specific to all the strains of Escherichis coli, of Shigella (with the exception of S. boydii serotype 13) and of Escherichia fergusonii. 
More particularly, this oligonucleotide is capable of hybridizing with the 637-660 region of the 16S RNA of E. coli (numbering system of Brosius et al., 1978). In effect, this portion of the 16S RNA is fairly well conserved in the Enterobacteria but not sufficiently to be specific to these. However, surprisingly, it turned out to be very interesting in that it allows the detection of the genomic species defined above without cross reaction with other species. The oligonucleotide according to the invention can likewise only hybridize specifically with at least 10 consecutive nucleotides of the 637-660 region of the 16S RNA of E. coli. In effect, with two oligonucleotides recognizing adjacent zones and then linked by a ligase, a longer oligonucleotide and therefore one which is more resistant to more stringent hybridization conditions is obtained.
It is thus possible to utilize two oligonucleotides representing the left half and the right half of one of the oligonucleotides of the invention, to make them hybridize with the target, to link them by the action of a ligase, and to increase the washing temperature so as to eliminate any small unlinked oligonucleotide. This method which reduces the background noise has been proposed by Alves and Carr (1988).
Advantageously, the oligonucleotide according to the invention corresponds to SEQ ID NO. 1.
In effect, an oligonucleotide of 24 nucleotides, complementary to the abovementioned 637-660 region of the 16S RNA of E. coli has been synthesized. It has been called Ec637 and identified SEQ ID NO. 1.
The labeling was obtained by a grafting of two chromophores (fluorescein or Texas Red) to each end of the oligonucleotide. The oligonucleotide probe used in in-situ hybridization at 42xc2x0 C. in the presence of 22% of formamide followed by washing at 60xc2x0 C. renders the cells of Escherichis coli, Shigella dysenteriae, Shigella flexneri, Shigella boydii (except the 13 serotype), Shigella sonnei and Escherichia fergusonii (E. coli genomic group) fluorescent. It does not react with the majority of the other species and genera tested. However, it has been observed that the species Citrobacter koseri and the species of Cedecea remain fluorescent after washing at 60xc2x0 C.
In fact, a temperature of the order of 61xc2x0 C. must be achieved in order that these species no longer react. However, at 61xc2x0 C., the E. coli genomic group is rendered very weakly fluorescent.
The present invention therefore likewise relates to an oligonucleotide allowing the obtainment of results which are even better in terms of specificity.
In effect, the Ec637 oligonucleotide was modified at the level of a nucleotide situated at a conserved (invariant) position of the corresponding 16S rRNA sequence in order to create a voluntary mispairing. This mispairing was carried out in the central part of the oligonucleotide. The sequence obtained was called Colinsitu and identified SEQ ID NO. 2. The introduction of a central mispairing has the aim of weakening the hybrid which is obtained. If a sequence differs by a sole nucleotide of the 637 to 660 sequence of E. coli, this will cause two mispairings with the Colinsitu probe which will not hybridize under the chosen experimental conditions. This probe remains reactive with respect to the E. coli genomic species and becomes inactive with respect to all the other species and genera. This specificity is maintained in a wide washing temperature range extending from 51xc2x0 C. to 59xc2x0 C.
The Colinsitu probe can be used in in-situ hybridization but likewise, in hybridization on a filter, in liquid medium, in reverse hybridization, or as a specific primer in a gene amplification system.
The invention likewise relates to complementary oligonucleotides of the oligonucleotides described below.
Other types of labeling of the probe (radioactivity, chemical or enzymatic labeling) can be used for hybridization in situ.
More particularly, the oligonucleotides according to the invention can be labeled at their 3xe2x80x2 or 5xe2x80x2 end or at the 3xe2x80x2 and 5xe2x80x2 ends.
The advantage of this probe, applied in in-situ hybridization with microscopic examination of the bacterial cells or detection by flow cytometry, is that it is able to detect, identify and count the cells of the genomic species E. coli in various samples such as clinical and veterinary samples (in particular urine), water and other drinks, food, the environment.
The invention likewise relates to a procedure for detection and visualization of bacteria of the genomic species Escherichis coli (including all the Shigellae with the exception of S. boydii serotype 13)/Escherichia fergusonii in a sample comprising a hybridization step of the ribosomal RNA of the bacteria of said genomic species with an oligonucleotide according to the invention, and more particularly with an oligonucleotide selected from SEQ ID NO. 1 and SEQ ID NO. 2.
More particularly, the hybridization in question can be an in-situ hybridization, a hybridization on a filter, a hybridization in liquid medium or a reverse hybridization.
Reverse hybridization for the purposes of the present invention is understood as meaning a hybridization reaction in which the oligonucleotide probe of interest is immobilized on a support, the nucleic acid to be detected and/or the organism containing the nucleic acid to be detected being present in solution.
According to a particular method of carrying out a reverse hybridization reaction according to the invention, the oligonucleotide probes can be employed within a detection device comprising a matrix bank of oligonucleotides. An example of production of such a matrix bank can consist of a matrix of oligonucleotide probes fixed to a support, the sequence of each probe of a given length being situated with a gap of one or more bases in relation to the preceding probe, each of the probes of the matrix arrangement thus being complementary to a distinct sequence of the target DNA or RNA to be detected and each probe of known sequence being fixed in a predetermined position on the support. The target sequence to be detected can advantageously be radiolabeled or nonradiolabeled. When the labeled target sequence is placed in contact with the matrix device, this forms hybrids with the probes of complementary sequence. Treatment with the nuclease, followed by washing, allows the target hybrid probes-sequences which are not perfectly complementary to be eliminated.
On account of the precise knowledge of the sequence of a probe at a determined position of the matrix, it is then possible to deduce the nucleotide sequence of the target DNA or RNA sequence and consequently to detect possible localized mutations in the ribosomal DNA of E. coli, and more particularly mutations affecting the 637-660 region of the DNA coding for the 16S rRNA of E. coli. 
One alternative to the use of a labeled target sequence can consist in the use of a support allowing a xe2x80x9cbioelectronicxe2x80x9d detection of the hybridization of the target sequence on the probes of the matrix support, when said support is formed of or comprises a material capable of acting, for example, as an electron donor at the positions in the matrix at which a hybrid has been formed. Such an electron-donor material is, for example, gold. The detection of the nucleotide sequence of the target DNA or RNA is then determined by an electronic device.
An example of production of a biosensor, such as defined above, is described in European Patent Application No. EP-0 721 016 (Affymax Technologies N.V.) or alternatively in American U.S. Pat. No. 5,202,231 (Crkvenjakov and Drmanac).
The invention likewise relates to the use of an oligonuleotide corresponding to SEQ ID NO. 1 or SEQ ID NO. 2 or differing from SEQ ID NO. 1 by a nucleotide or a complementary oligonucleotide as a primer for carrying out a gene amplification procedure, such as PCR.
The oligonucleotides according to the invention can likewise be used in a hybridization inhibition method. In effect, it is possible to envisage fixing to a support (filter, cupule or microchip) an oligonucleotide which is identical or homologous to the 637-660 region of the 16S RNA of E. coli and to label in any manner an oligonucleotide which is complementary to this region according to the present invention. In the absence of competitor DNAs or RNAs, the two oligonucleotides have to reassociate completely. The introduction into the system of a nucleic acid capable of reassociating with one or other of the nucleotides (for example a nucleic acid belonging to one of the species aimed at by the present invention) or both (case of two separate strands) inhibits the fixing of the oligonucleotide, which is free, labeled and according to the invention, to the support.
The present invention likewise relates to a procedure for detection and visualization of microorganisms by hybridization allowing the specificity of the oligonucleotide probe used to be optimized. In effect, an oligonucleotide is all the more specific the more net differences it has in its hybridization capacities with, on the one hand, the target sequences and, on the other hand, the other sequences. Under the experimental hybridization conditions, this difference is all the more detectable the more sequence differences (or mispairing) there are between the abovementioned oligonucleotide and the sequence with which it is capable of hybridizing. Consequently, it becomes advantageous to artificially increase the number of these mispairings by modifying the oligonucleotide used for the hybridization at the level of a nucleotide which is generally highly conserved at the level of the sequence which it is sought to detect.
Consequently, the present invention relates to a procedure for detection and visualization of microorganisms (or of a group of microorganisms) by hybridization employing an oligonucleotide which is complementary to the target sequence of the microorganism with the exception of a nucleotide located in the central part of said oligonucleotide. The nucleotide in question is located in an invariant position of the target sequence of the microorganisms and is preferably in a central position.
For example, for a complementary oligonucleotide with a length of 20 base pairs, the noncomplementary nucleotide is located between positions 7 and 13 inclusive according to a numbering of the oligonucleotide commencing at its N-terminal end, preferably, the nucleotide in question is located at position 10.
The invention therefore relates to a procedure for detection and visualization such as described above applied to bacteria of the genomic species Escherichia coli (including all the Shigellae with the exception of S. boydii serotype 13)/Escherichia fergusonii. 
Thus, in the context of the present invention, the complementary oligonucleotide employed in the abovementioned procedure is an oligonucleotide according to the present invention and differing from SEQ ID NO. 1 only by one oligonucleotide and preferably corresponding to SEQ ID NO. 2.
Among the potential applications of the invention, the following will be mentioned more particularly:
Search for confirmation that the atypical strains of E. coli definitely belong to this species. The Reference Centers often receive strains which could be atypical E. coli. They give unusual biochemical reactions for this species such as negative reaction for the production of indole or of gas, the hydrolysis of o-nitrophenol-xcex2-galactopyranoside, the hydrolysis of beta-glucuronides, unusual fermentation reactions, or very poor growth in the usual media. The Colinsitu probe can confirm whether these strains belong to the genomic species E. coli-E. fergusonii. If the reaction with the probe is positive, it is easy to distinguish E. fergusonii by the fermentation of adonitol and of cellobiose;
detection, identification and counting of E. coli in the urine of sick people or infected animals. The majority of urinary infections being due to E. coli and a urinary colonization or infection being characterized by the presence of more than 1000 or 10,000 bacteria per ml, hybridization in situ with Colinsitu of an appropriate dilution of urine should allow the presence of E. coli to be confirmed and for it to be counted in the urine in 2 to 3 hours;
detection and counting of E. coli in water and food. Escherichis coli is the principal biological indicator of fecal contamination of water and of food. It suffices to filter a known and sufficient quantity of water and to carry out the hybridization in situ on the filters thus obtained. If, with the aid of a micrometer and a reticule, the filtered volume related to a surface observed in the microscope is known, it is possible to quantify the number of cells of E. coli in the water. In the case of foods which cannot be filtered and which must not have one E. coli per 25 g, enrichment can be necessary starting from 25 g of food; in situ hybridization then carried out on the culture medium will indicate if E. coli is present.
The invention is not limited to the above description but encompasses all the variants thereof. The examples below allow it to be better understood while only being mentioned in purely by way of illustration.